1. Field of the Invention
The present invention demonstrates the method of quantitative analysis for thuringiensin by capillary electrophoresis (CE) and use Tryptophan as an internal standard.
2. Description of the Prior Art
Thuringiensin is the .beta.-exotoxin secreted by Bacillus thuringiensis, and is one kind of nucleotide analogue, it's poison against insects and Nematoda, mite and pertains to a broad spectrum of biological insecticide. There is a large potential market. So far there are some breakthrough in fermentation and recovery technology. However, so far there are no accurate, rapid and effective method of quantitative analysis. HPLC has replaced the prior bioassay as quantitative assay is time-consuming and labor intensive.
HPLC (high performance liquid chromatography) has been used to detect Bacillus thuringiensis ever since 1982. In the beginning the technology is not mature such that it lacks specificity to complicated mixing of fermentation solution and the sensitivity is low. Then buffer gradient elution technique was used for improvement, but it's still time-consuming. Recently people found that the concentration of thuringiensin is proportional to the peak area in the eluting buffer which contains 50 mM Di-hydrogen potassium phosphate at 2.8 PH. And it takes only 20 minutes to complete the assay. The HPLC analysis of various concentrations of standards has shown that the concentration of thuringiensin is linearly proportional to the peak area. And the resultant chromatogram is quite repeatable and has good specificity. Therefore, it's an inevitable tendency that HPLC will replace the conventional bioassay.
There are still some disadvantages although HPLC is efficient. For example, it has to stabilize the baseline before the assay is started, and also the column is expensive with a limited lifetime. Recently, there is a new CE technique which can separate different molecules more rapidly, more accurately and can do qualitative & quantitative analysis. New CE techniques can speed up the analyzing time, simplify the analyzing process, and can make the analysis more efficient and accurate. For quantitative analysis of thuringiensin, a purified standards is required as reference. It's quite difficult to purify Thuringiensin due to the low molecular weight (MW=701) and low concentration in fermentation broth. In addition, the purified thuringiensin is unstable and thus becomes difficult to preserve. The present invention-Tryptophan or some Amino Acids such as tyrosine or phenylalanine are used as an internal standard, to replace the purified Thuringiensin for quantitative analysis.